摘要
目的研究低氧/HIF-1α通路对RAW264.7细胞向成熟破骨细胞分化的调控作用,并初步探讨其可能的分子机制。方法采用短发夹RNA(short hairpin RNA,shRNA)慢病毒感染法,分别建立HIF-1α和NRP-1稳定低表达的RAW264.7细胞系。将RAW264.7细胞分组培养:对照组,分化组(RANKL 100 ng/mL+50%成骨细胞CM),缺氧组(RANKL 100 ng/mL+50%成骨细胞CM+100μmol/L CoCl_2氯化钴),缺氧+HIF-1α-NC组,缺氧+HIF-1α-shRNA干扰组,缺氧+NRP-1-NC组及缺氧+NRP-1-shRNA干扰组。TRAP染色观察破骨细胞的分化情况;RT-PCR检测Cath K、MMP-9、TRAP、HIF-1α和NRP-1 mRNA的表达;Western blot检测HIF-1α和NRP-1蛋白的表达。结果①缺氧组破骨细胞分化相关基因Cath K、MMP-9、TRAP mRNA的表达量及TRAP染色阳性细胞数均明显低于分化组(P<0.05)。②缺氧组细胞中HIF-1α和NRP-1的表达均明显高于分化组(P<0.05)。③与缺氧组相比,缺氧+HIF-1α-shRNA干扰组TRAP染色阳性细胞数及破骨细胞分化相关基因的表达均明显增加(P<0.05);且HIF-1α和NRP-1表达均明显减少(P<0.05)。④与缺氧组相比,缺氧+NRP-1-shRNA干扰组TRAP染色阳性细胞数及破骨细胞分化相关基因的表达均明显增加(P<0.05);且NRP-1表达明显减少(P<0.05)。结论在RAW264.7细胞中,CoCl_2诱导的缺氧能够通过上调HIF-1α增强NRP-1的表达,从而抑制其向成熟破骨细胞分化。
Objective To investigate the regulation effect of hypoxic/HIF-1α pathway on the differentiation of RAW264.7 cells into mature osteoclasts, and to explore the possible molecular mechanism. Methods RAW264.7 cell lines with stable low expression of HIF-1α and NRP-1 were respectively established by short hairpin RNA(shRNA) lentivirus infection. Then the RAW264.7 cells were cultured in groups, that is, control group, differentiation group [100 ng/mL RANKL+50% osteoblast conditioned medium(CM)], hypoxia group(100 ng/mL RANKL+50% osteoblast CM+100 μmol/L CoCl2), hypoxia+HIF-1α-NC group, hypoxia+HIF-1α shRNA group, hypoxia+NRP-1-NC group, and hypoxia+NRP-1-shRNA group. TRAP staining was used to observe osteoclast differentiation. The mRNA expression of Cath K, MMP-9, TRAP, HIF-1α and NRP-1 were detected by RT-PCR. Western blotting was used to detect the expression of HIF-1α and NRP-1 proteins. Results ① The mRNA expression levels of Cath K, MMP-9 and TRAP and the number of TRAP positive cells in the hypoxia groups were significantly lower than those in the differentiation groups(P<0.05).② The expression levels of HIF-1α and NRP-1 were significantly higher in the hypoxic groups than the differentiation groups(P<0.05).③ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while those of HIF-1α and NRP-1 were significantly reduced in the hypoxic+HIF-1α shRNA group(P<0.05).④ Compared with the hypoxia groups, the number of TRAP positive cells and the expression levels of osteoclast differentiation related genes were significantly increased, while the level of NRP-1 was significantly decreased in the hypoxia+NRP-1-shRNA group(P<0.05). Conclusion The hypoxia-mediated HIF-1α-dependent up-regulation of NRP-1 inhibits the differentiation of RAW264.7 cells into mature osteoclasts.
作者
李松涛
孙靖
陈武桂
马敏
张莹
牛晓健
周驰雨
初同伟
LI Songtao;SUN Jing;CHEN Wugui;MA Min;ZHANG Ying;NIU Xiaojian;ZHOU Chiyu;CHU Tongwei(Department of Orthopaedics,Second Affiliated Hospital,Army Medical University ( Third Military Medical University),Chongqing,400037,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第11期1052-1058,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81570800)~~